The present invention relates to a novel method for enzymatic preparation of homogentisate, or 2,5-dihydroxyphenylacetic acid (hereafter HMO).
HMO is a known precursor of molecules termed ochronotic pigments which are melanin analogues. These brown molecules are, moreover, often cited as xe2x80x9cmelanin-like pigmentsxe2x80x9d which find a varied application in cosmetics or the pharmaceutical industry. The addition of melanin or melanin-like pigments to antisun milks would have an advantageous protective effect. To produce these ochronotic derivatives from HGA, the method is simple since the molecule self-oxidizes rapidly under alkaline conditions. Various methods for enzymatic preparation of HMO from 1-phenylacetic acid or from tyrosine are described in the prior art (WO 93/08295 or EP 343 330), as well as the subsequent preparation of melanin.
It is also known that HMO is a compound which is essential to plant life. In plant cells, HMO is the product of enzymatic transformation of 4-hydroxyphenylpyruvate (hereafter HPP) with 4-hydroxyphenylpyruvate dioxygenase (hereafter HPPD). Inhibitors of this enzyme are herbicidal compounds which block the production of HMO in plant cells (Pallett K. E. et al. 1997 Pestic. Sci. 50 83-84). When plants of Arabidopsis thaliana for example, are germinated on synthetic medium in the presence of an HPPD inhibitor, the plants will germinate, remain white and then die very rapidly. However, if HMO is added to the synthetic medium supplemented with an HPPD inhibitor, the plants will germinate normally and remain green as long as the medium contains HMO. It is thus also important to have HMO available to prevent deficiencies in plants which are linked to a natural or induced, in particular by HPPD inhibitors, metabolic dysfunction of HMO biosynthesis.
The present invention thus relates to a method for enzymatic preparation of HMO from HPP, and more particularly to a method for enzymatic preparation of HMO from HPPD-inhibitor-insensitive HPP.
The method according to the invention consists in carrying out, in a suitable reaction medium, the following enzymatic reactions:
enzymatic conversion of HPP into 4-hydroxyphenyl-acetate (hereafter HPA) with a first suitable enzyme, then
enzymatic conversion of HPA into HMO with a second suitable enzyme.
The following first enzymatic reaction
4-hydroxyphenylpyruvate (HPP)xe2x86x924-hydroxyphenylacetate (HPA)
is catalysed by a suitable HPP-oxidase. Such oxidases are found in many prokaryotic or eukaryotic species, in particular in bacteria which can grow on HPP as the only carbon source, transforming it into HPA, more particularly in an Arthrobacter in which such an oxidase is responsible for a step in tyrosine catabolism (Blakley, E. R. 1977 Canadian Journal of Microbiology 23 1128-1139).
The following second enzymatic reaction:
4-hydroxyphenylacetate (HPA)xe2x86x92homogentisate (HMO)
is catalysed by a suitable HPA-hydroxylase. Such hydroxylases are found in many prokaryotic or eukaryotic species, in particular in bacteria which can grow on HPA as the only carbon source, transforming it into HMO, more particularly in Pseudomonas acidovorans, often termed Comamonas acidovorans (Hareland, W. A. et al 1975 Journal of Bacteriology 121 272-285), in certain Xanthobacter (Van Den Tweel W. J. J. et al. 1986 Antonie val Leeuwenhoek 52 309-318), in Pseudomonas alcaligenes (Karigar C. S. and Pujar B. G. 1993 FEMS Microbiology Letters 110 59-64), in Flavobacterium sp. (Van Den Tweel W. J. J. et al. 1988 Arch Microbiol. 149 207-213), in Bacillus subtillis (Crawford R. L. 1978 FEMS Microbiology Letters 4 233-234), in Nocardia sp. DM1 (Raju S. G. and Vaidyanathan C. S. 1986 J. Indian Inst. Sci. 66 511-520) and in Rhodococcus erythropolis (Suemori A. et al. 1996 Journal of Fermentation And Bioengineering Vol. 81, No. 2 133-137).
The HPA-hydroxylase used in the method according to the invention is advantageously extracted from Pseudomonas acidovorans. 
According to a preferential embodiment of the invention, both enzymatic reactions are carried out in the same reaction medium containing HPP, the two suitable enzymes being present together at the same time in the reaction medium.
The two suitable enzymes can be introduced into the suitable reaction medium in the form of protein extract, said protein extract being able to be crude or totally or partially purified, or alternatively they can be produced in situ by suitable biological organisms. They can thus be produced in situ by each biological organism which naturally produces the two enzymes, or alternatively by a single biological organism which has been modified so as to produce the two enzymes. This biological organism can be a bacterium, a yeast or a plant cell.
Since the two enzymes are insensitive to HPPD inhibitors, the method according to the invention can be performed in the presence of an HPPD inhibitor in the suitable reaction medium.
The suitable reaction medium consists of any aqueous medium in which the temperature, pH and ionic strength conditions are suitable for the enzymatic reactions. When the enzymes are produced in situ by one or more biological organisms, the reaction medium is suitable for the growth of said organisms.
At the end of the reaction, HMO can be isolated from the reaction medium and purified, or left in the reaction medium. In this second case, the reaction medium containing HMO can be used as a nutrient medium for culturing plants, more particularly for culturing plants which exhibit a natural or induced, in particular by HPPD inhibitors, metabolic dysfunction of HMO biosynthesis.
The examples below make it possible to illustrate the invention, without however seeking to limit the scope thereof.